Topical formulation for prevention and treatment of acne

ABSTRACT

Disclosed is a topical formulation for preventing or treating acne, in particular to a topical formulation for preventing or treating acne through the antimicrobial activity of the formulation against acne-causing bacteria,  Propionibacterium acnes , inhibition of excess production of sebum by inhibition of excess production of sebum by inhibition of 5α-reductase, inhibition of comedo, keratolysis and anti-inflammatory action, which comprises extract obtained from at least one oriental medicine selected from the group consisting of  Cavalia gladiata, Biota orientalis  and  Coptis chinensis.

This application is a 371 of PCT/KR2003/001626 filed on Aug. 12, 2003,published on Feb. 26, 2004 under publication number WO 2004/016239 A1which claims priority benefits from South Korean Patent ApplicationNumber 10-2002-0048073 filed Aug. 14, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a topical formulation for preventing ortreating acne through antimicrobial activity against acne-causingbacteria, Propionibacterium acnes, inhibition of excess production ofsebum by inhibition of 5α-reductase, inhibition of comedo, keratolysisand anti-inflammatory action, which comprises extract obtained from atleast one oriental medicine selected from the group consisting ofCavalia gladiata, Biota orientalis and Coptis chinensis.

2. Description of the Related Technical Field

Acne is developed in teen-agers at an incidence of 90% but also found inadults of in their twenties- or thirties at rare intervals. Acne (oracne vulgaris) is a chronic inflammatory disease or a disorder developedat sebaceous gland and hair follicle, of which etiopathology includesexcess secretion of sebum, dyskeratinization of epidermis of hairfollicle, overgrowth of anaerobic skin-colonizing bacterium,Propionibacterium acnes and other causes. Acne is generally found onface, chest, back, neck and brachium, the most noticeable parts of skin,and characterized as comedo, pustule, lupus, petty knob or scar.

At the early stage of acne, the excessively-secreted sebum isaccumulated in hair follicle and the hair follicle is in turn swelleddue to pressure of sebum, resulting in the occurrence of comedo, whereinthose with closed hair follicle become white acnes (closed comedo) whilethose with slight-opened hair follicle tip become black acnes (opencomedo). Upon further development of acne, bacteria elicit inflammationthat contributes to the occurrence of two different types of symptoms: apapule type showing reddish skin with petty comedo and a pustule typeshowing pus. There are a few differences between acnes of teen-agers andadults, as summarized in Table 1.

TABLE 1 Differences between Acnes of Teen-agers and AdultsClassification Teen-ager acnes Adult acnes Location of Brow, entireface, any Tend to position at the lower Occurrence parts where sebaceouspart of face such as cheek, gland exists girth of mouth and jaws ascompared to teen-agers Season of Spring and Summer in Year-roundoccurrence Occurrence association with without seasonal outbreakactivation of sebaceous gland Symptoms Developed Developedsimultane-ously simultaneously with with skin dryness, repetition excesssebum, of petty acne boss (shot appearance of appearance type), slowpetty comedo at the progress in treatment, vestige activated part ofsebum of acnes remained secretion Causes Physical instability Closelyrelated to physical during growth period and mental conditions of bodysuch as hormonal imbalance, insufficient sleep, fatigue, stress andmenstruation. Low relation to skin conditions

As shown in Table 1, the teen-ager acnes are developed at the entireparts of brow and face in spring and summer when the secretion of sebumis active and they are very likely to develop in oily skins. Incontrast, the adult acnes are apt to develop throughout the year atrestricted parts and are known to have little or no relation with lipidcontent. The development is affected by physical and mental conditionsof body such as hormonal imbalance, stress, irregular dietary habits andpoor body conditions thus implying complexity of its etiology. Evenafter fairly good treatment of adult acnes, it is very likely thatpigmentation occurs to produce freckles and also the affected skinsurface is sometimes caved in to form acne crater at the site. Thehormones that have drawn much attention as known to cause adult acnesare androgen, which secretes sebum through stimulation of sebaceousgland and estrogen, the antagonist of androgen. Where the balance of twohormones is disrupted, it results in secretion of excess sebum and acneis developed as a result.

A variety of pharmaceuticals have been developed as an effort to treatacnes, which include antibiotics such as erythromycin andbenzoylperoxide to inhibit a proliferation of acne-causing bacteria andestrogen to regulate sebum secretion, however, most of which aregenerally associated with adverse effects. In cosmetics, vitamin Aderivatives to remove keratin; and trichloric acid and salicylic acid toinhibit a proliferation of acne-causing bacteria have been developed andtested. However, these active ingredients exhibit a little effect butare usually associated with adverse effects such as skin flaring, skinhypersensitivity and light hypersensitivity. Further, the recurrence ofacnes are usually observed when administered with these activeingredients. Consequently, there are not genuine products capable ofpreventing and treating acne.

SUMMARY OF THE INVENTION

In an effort to develop a novel topical formulation for treating orpreventing acnes, the present inventors have performed extensiveexperiments using active ingredients allegedly known to be involved inmechanism of acne treatment (antimicrobial activity, inhibition ofexcess production of sebum, keratolysis, anti-inflammatory action, etc.)based on the disclosures in various publications related to orientalmedicines. As a result, the present inventors have found that Cavaliagladiata, Biota orientalis and Coptis chinensis are very effective inprevention and treatment of acnes and also have very little or noirritation.

Accordingly, it is an object of this invention to provide a topicalformulation for preventing or treating acnes, which comprises extractobtained from at least one oriental medicine selected from the groupconsisting of Cavalia gladiata, Biota orientalis and Coptis chinensis.

It is another object of this invention to provide a composition oforiental medicine to exhibit therapeutic effects on acnes with little orno irritation.

DETAILED DESCRIPTION OF THIS INVENTION

The present invention is directed to a topical formulation forpreventing or treating acnes, which comprises extract obtained from atleast one oriental medicine selected from the group consisting ofCavalia gladiata, Biota orientalis and Coptis chinensis.

The present invention will be described in more detail hereunder:

The present invention relates to a topical formulation for preventing ortreating acne through antimicrobial activity against acne-causingbacteria, Propionibacterium acnes, inhibition of excess production ofsebum by inhibition of 5α-reductase, inhibition of comedo formation,keratolysis and anti-inflammatory action, which comprises extractobtained from at least one oriental medicine selected from the groupconsisting of Cavalia gladiata, Biota orientalis and Coptis chinensis.

Cavalia gladiata is an annual plant and belongs to Leguminosae. Sinceits bean hull looks like a straw cutter, it is generally called a swordbean. It has been generally cultivated in the southern part of Korea andthe region of Jangriver and the southern parts of China. Its root andhusk is also used as medicines. It has been known that Cavalia gladiatacontains Vitamins A and C, a hydrolytic enzyme urease, hemmagglutinineand canavanine. It has been used since old times as raw materials forhigh-quality food and oriental medicine in China such as medicines fortreating hiccough due to cold sweat, vomiting and stomachache.Currently, in the field of folk remedy, Cavalia gladiata is known to beeffective in treatment of purulent diseases such ozena and hemorrhoids.

Coptis chinensis is a perennial herbaceous plant that belongs to thefamily of a buttercup and its root stem has been used from the beginningas a raw material for oriental medicines. It has been known as a safeplant because it elicits no irritation to skin and mucous membrane.Coptis chinensis has been reported to contain alkaloids such asberberine, coptisine, worenine, palmatine and columbamaine together withobakunone and obakulactone. It has been used in China to treat scald,purulent infection, infectious dermatis, ozena, palatum inflammation andexudative erythema multiforme, and used as raw materials of medicinesfor gastrointestine and intestinal disorders.

Biota orientalis is a seed from a thuja that belongs to Cupressaceae.The fresh ones look light yellow or yellowish white and old ones lookyellowish brown and leak oil. Usually, Biota orientalis has beencultivated in various regions of China and its seed contains about 14%oil and a small amount of saponin. It has been used for treatinganxieties and astriction and also as a raw material for medicines totreat inflammation and depilation as well as a calmative.

The extract of this invention may be prepared by the steps ofpowderizing Cavalia gladiata, Biota orientalis and Coptis chinensis,respectively, heat-extracting the powder in at least one extractionsolvent selected from the group consisting of distilled water, methanol,ethanol, propanol, butanol, glycerol, propyleneglycol, 1,3-butyleneglycol, methyl acetate, ethyl acetate, benzene, hexane, diethyl etherand dicholoromethane, filtering the extract, concentrating and freezedrying the resultant under reduced pressure.

The mixed extract of Cavalia gladiata, Biota orientalis and Coptischinensis is prepared by powderizing Cavalia gladiata, Biota orientalisand Coptis chinensis, respectively, mixing the powders and extractingthe mixed powders; or powderizing Cavalia gladiata, Biota orientalis andCoptis chinensis, respectively, extracting each powder and mixing theextracts.

When a mixed extract is used, the formulation of this inventioncomprises the extract from two different kinds of oriental medicines,where each extract comprises 0.01-10 wt % of Cavalia gladiata and0.01-10 wt % of Biota orientalis; 0.01-10 wt % of Cavalia gladiata and0.001-5 wt % of Coptis chinensis; or 0.01-10 wt % of Biota orientalisand 0.001-5 wt % of Coptis chinensis. In addition, the formulationcomprises the extract obtained from three different kinds of orientalmedicines, where each extract comprises 0.01-10 wt % of Cavalia gladiataand 0.01-10 wt % of Biota orientalis; and 0.001-5 wt % of Coptischinensis. If the concentration is lower than the above range, theefficacy becomes negligible; in the case of exceeding, adverse effectson skin and the technical problems associated with formulation occur.

It is preferred that the extract from at least one oriental medicineselected from the group consisting of Cavalia gladiata, Biota orientalisand Coptis chinensis is contained in an amount of from 0.001 wt % to20.0 wt %. If the amount is less than 0.001 wt %, the treatment efficacyis very poor; in the case of exceeding 20.0 wt %, adverse effects onskin and the technical problems associated with formulation occur.

The formulation of this invention may be in the form of emulsion, gel,pack, cosmetic liquid or soap for cosmetics, or ointments or patches forpharmaceuticals along with a pharmaceutically acceptable carrier or avehicle. Each particular formulation may be prepared in accordance withthe conventional processes.

The effective dose of the present formulation for topical administrationmay vary depending on shape, size, part of occurrence and age of asubject, and it is generally applied twice to several times a day.

The topical formulation of this invention comprising at least oneoriental medicine selected from the group consisting of Cavaliagladiata, Biota orientalis and Coptis chinensis is very effective in theprevention and treatment of acnes.

The following specific examples are intended to be illustrative of theinvention and should not be construed as limiting the scope of theinvention as defined by appended claims.

EXAMPLE 1 Preparation of Extract from Cavalia gladiata, Biota orientalisand Coptis chinensis

The extracts obtained from Cavalia gladiata, Biota orientalis and Coptischinensis used in this invention were prepared as follows:

200 g each of Cavalia gladiata, Biota orientalis and Coptis chinensiswere washed with distilled water, dried and powderized with a pulverizerspecially designed for oriental medicine, and to the resultant about10-fold volume of 80% ethanol solvent were added. The extraction wasperformed by heating for 8 hr 80° C. using a device equipped with acooling condenser to prevent the evaporation of a solvent. The extractwas filtered through a 400-mesh filter cloth and the remainder wasfiltered once. Each extract from oriental medicines was cooled to roomtemperature and filtered through Whatman No. 2 filter paper, and thefiltrate was concentrated at 48° C. under reduced pressure with aretrieving solvent evaporated by use of a distilled apparatus equippedwith the cooling condenser (Buchi Rotavapor R-124, Water Bath B-480 Madein Switzerland, Eyela A-3S Tokyo rikakikai), followed by freeze drying,finally yielding 15.8 g of Cavalia gladiata extract, 8.9 g of Biotaorientalis extract and 14.6 g of Coptis chinensis extract as dry weight.

EXAMPLE 2 Evaluation of Inhibitory Effect on 5α-Reductase

Testosterone 5α-Reductase, used to examine the inhibitory effect of theextract obtained from oriental medicines against testosterone5α-reductase activity, was prepared at a temperature of below 4° C. Thewhite female Sprague-Dawley rat aged at 8 weeks was anesthetized byinhalation of diethylether and sacrificed by the disjunction ofcervicals, followed by liver ablation. The extracted liver was washedthree times with PBS (phosphate buffered saline) and 3 volumes ofice-cold buffer (50 mM sodium phosphate, pH 6.8, 0.25 M sucrose, 1 mMdithiothreitol) were added, followed by pulverizing with a cellhomogenizer (Polytron Homogenizer), obtaining homogenized suspension.For isolating 5α-Reductase from the suspension thus obtained, thesuspension was subject to ultrasonication for 5 min and centrifugation(15,000 rpm, 4° C., 5 min) for preparing a protein layer. Thesupernatant was resuspended in the buffer described above and stored ata temperature of −80° C. The protein quantification of the proteinsuspension obtained thus was performed according to Bradford method andits accurate value was determined with ELISA reader.

For evaluating the inhibitory effect on the activity of 5α-Reductase, to50 μl of the reaction solution, 10 μl of ethanol solution of inhibitoror tested sample (70% ethanol solution containing extract from orientalmedicine) were added and the protein suspension was added to the volumeof 100 μl of the reaction solution. The reaction solution contains 50 mMNa₂HPO₄ (pH 6.8), 25 mM KCl, 500 mM NADPH and 50 mM (3H) testosterone.The reaction mixture was reacted in water bath for 10 min at 37° C. and250 μl of the reagent for reaction termination (mixture of 70%cyclohexane and 30% ethylacetate) were added to terminate the reaction.To obtain steroids from the reactant terminated, the reactant wascentrifuged to prepare supernatant and the supernatant was placed inhood for 1 day for evaporating the reaction solvent, after which theremainder obtained was dissolved in 20 μl of chloroform. The resultantwas spotted on TLC plate and developed for 30 min with a developingsolution (toluene:acetone=4:1) in TLC chamber and the plate developedwas exposed for 3 days to hyperfilm. The area exposed was measured withdensitometer and the inhibitory rate against testosterone 5α-Reductasewas calculated as shown in the below formula 1. The results aresummarized in Tables 2 and 3.Inhibitory rate against testosterone 5α-reductase(%)=[(A−B)/A]×100  Formula 1

wherein A is the conversion rate of testosterone to dihydro-testosteronewith no addition of a sample, and B is the conversion rate oftestosterone to dihydro-testosterone with the addition of a sample.

TABLE 2 Inhibitory rate against 5α-reductase Active ingredients Conc.(%) (%) Cavalia gladiata 0.1 100.0 0.01 38.3 Biota orientalis 0.1 100.00.01 94.0 0.001 34.7 Coptis chinensis 0.1 100.0 0.01 44.9 Azelaic acid1.0 100.0 0.1 19.8

TABLE 3 Inhibitory rate against 5α-reductase depending on conc. (%)Items 0.1% 0.05% 0.01% Biota 100 100 35.2 orientalis:Cavaliagladiata(50:50) Biota orientalis:Coptis 100 100 46.9 chinensis(50:50)Cavalia gladiata:Coptis 100 100 31.7 chinensis (50:50) Cavaliagladiata:Biota 100 100 66.8 orientalis:Coptis chinensis (34:33:33)Cavalia gladiata:Biota 100 100 92.6 orientalis:Coptis chinensis(70:20:10)

As shown in Table 2, each of three different types of extract (Cavaliagladiata, Biota orientalis and Coptis chinensis) is revealed to exhibitthe inhibitory effect against the activity of 5α-Reductase. In addition,when at least two different kinds of extracts were mixed at a suitableratio and dissolved in 70% ethanol to make 0.1, 0.05 and 0.01% (w/v) ofextracts, the extract to be examined showed a significantly higherinhibitory effect against the activity of 5α-Reductase even at a lowconcentration as shown in Table 3, compared to that of extractcontaining single kind of extract. These results represent that themixed extract at a suitable ratio shows a synergistic inhibitory effectagainst the activity of 5α-Reductase. It would be understood by oneskilled in the art that the present compositions exhibiting asynergistic inhibitory effect against the activity of 5α-Reductase arenot limited to those indicated previously.

EXAMPLE 3 Evaluation of Antimicrobial Activity Against Acne Pathogen

The examination on antimicrobial activity of three extracts (Cavaliagladiata, Biota orientalis and Coptis chinensis) was performed in such amanner that an acne pathogen (Propionibacterium acnes) that was culturedin liquid medium was treated with a predetermined concentration ofextract powderized by freeze drying and then cultured in an anaerobicincubator, followed by measuring a minimum inhibitory concentration(MIC) against an acne pathogen (Propionibacterium acnes). The overallprocedure is as follows:

The acne pathogen (Propionibacterium acnes) was cultured at 37° C. for 3days in reinforced clostridial medium and then continued to subcultureuntil showing significant activity.

First, in a liquid culture method, 10 ml of reinforced clostridialmedium were introduced to screw cap tube and sterilized for 20 min at121° C. After sterilization, 0.5 ml of liquid paraffin sterilizedseparately was added dropwise on the liquid medium to protect thecontact with oxygen. After cooling the medium, the microbe wasinoculated into the medium and cultured at 37° C. for above 72 hr.

Second, in a solid culture method, 10 ml of the solid medium prepared byadding 0.1% agar to reinforced clostridial medium was introduced to ascrew cap tube and sterilized at 121° C. for 20 min. After cooling themedium, the microbe was inoculated into the medium by use of inoculatingplatinum loop and stab-cultured for 72 hr at 37° C.

Third, in a plate culture method, the agar medium prepared andsterilized as a solid culture method was poured to a plate and cooled.After cooling and solidifying completely, the plate was placed in ananaerobic jar, 10 ml of water were introduced to complex gas (CO₂ 10.5%,H₂ 9.6%, N₂ 79.9%) and the lid was kept covered, followed by culturingat 37° C. for 72 hr.

1 ml of the medium containing Propionibacterium acnes subjected to asufficient subculture was taken and inoculated into a medium containing9 ml of reinforced clostridial medium, thereby obtaining 10-fold dilutedculture medium. The same procedure was repeated to obtain between 10⁻¹and 10⁻⁵4-fold diluted culture media. Each of the homogeneous solutionscontaining Cavalia gladiata, Biota orientalis, Coptis chinensis and atleast two combinations thereof was well dissolved in 10 ml of 70%ethanol to obtain 2-fold diluted solution. The solution was filtered forremoving bacteria through 0.2 μm membrane filter. The reinforcedclostridial-agar medium was sterilized and cooled to 50° C. and to it,the extract removed of bacteria by filtering was added to aconcentration of 10 μg/ml-0.1 mg/ml. 1 ml of the diluted medium (mediumof Propionibacterium acnes subject to subculture diluted to 10⁻¹-10⁻⁵)was dispensed in a petri dish and each agar medium having certainconcentration was dispensed, followed by mixing and keeping it to standfor solidifying the agar medium. After solidification, the medium wasplaced in an anaerobic jar that in turn was equipped with gas-pak.Following culturing at 37° C. for 3 days, the number of colonies formedwere counted and compared to that of a control group, and the inhibitoryrate was calculated based on the results. The inhibitory rates againstP. acnes are shown in Tables 4 and 5.

TABLE 4 Concentration of extract (%) MIC to P. acnes 0.50 0.10 0.05 0.030.01 0.005 Cavalia gladiata −* − − − ± + Biota orientalis − − − ± + +Coptis chinensis − − − − − ± Benzoylperoxide − ±** +*** + + +*inhibition of growth **pseudo-positive (

) ***positive

As shown in Table 4 demonstrating the antimicrobial activity against P.acnes, the antimicrobial activity could be observed at a lowconcentration (0.03%) of extract and the acne-causing bacteria wascompletely killed at a high concentration (above 0.1%), while eachextract showed a little difference in its MIC against P. acnes.Therefore, it would be appreciated that the extract from orientalmedicine of this invention shows a noticeable bacteriocidal effect on P.acnes even at an extremely low concentration.

TABLE 5 Antimicrobial activity according to various concentrations (%)Extracts 0.1% 0.05% 0.01% 0.005% Biota orentalis:Cavalia 100 100 85.733.2 gladiata(50:50) Biota orientalis:Coptis 100 100 89.8 44.9chinensis(50:50) Cavalia gladiata:Coptis 100 100 92.3 49.7chinensis(50:50) Cavalia gladiata:Biota 100 100 92.4 46.8orientalis:Coptis chinensis(34:33:33) Cavalia gladiata:Biota 100 10092.6 48.6 orientalis:Coptis chinensis(70:20:10)

As shown in the results, the composition comprising at least twoextracts obtained from Cavalia gladiata, Biota orientalis and Coptischinensis at a certain ratio showed slightly increased antimicrobialactivity compared to that comprising a single extract. In particular,the composition containing three different extracts obtained fromCavalia gladiata, Biota orientalis and Coptis chinensis at a certainratio was observed to exhibit excellent antimicrobial activity andinhibitory effect against 5α-reductase at each concentration.

EXAMPLE 4 Evaluation of Inhibitory Activity to Hypercornification

All skin cells help to maintain skin homeostasis through suitablemetabolic processes by controlling the rate of their proliferation andexfoliation. Therefore, the reproduction rate of skin could beindirectly examined by observing the exfoliation rate of stratum corneumthat can be generally measured by evaluating the level of skindiscoloration associated with keratolysis.

In evaluating the efficacy of extracts, the first screening forpromoting keratolysis was performed using hairless mouse. Each extractsample was prepared by dissolving the dried powder extract prepared inExample 1 in 70% ethanol and the positive controls including glycolicacid or lactic acid were prepared as described above. The experimentswere carried out as follows:

The skin of a hairless mouse was washed with 10% SDS for 30 min and thenallowed its skin to dry for experiment. 0.4 ml of aqueous silver acetateliquid was covered with a hill top chamber and closed patch wasperformed for 30 min, followed by infiltrating with a photo-developingsolution to blacken a portion of patch with silver acetate. The closedpatch was further performed with each extract sample on the blackenedportion and removed after about 24 hr. The coloring index beforeapplication of extract and discoloring index at 24 hr after removal weremeasured by means of calorimeter (The higher the discoloring index, thehigher is the keratolysis effect)

TABLE 6 Conc. of Extract Appearance of applied (%) Sample Exfoliation0.2 0.1 0.05 Cavalia Homogeneous ++¹⁾ + + gladiata Biota orientalisHomogeneous +²⁾ ±³⁾ −⁴⁾ Coptis Homogeneous ++ ± − chinensis Glycolicacid Heterogeneous 2 wt % (pH 4) ++ Lactic acid Heterogeneous 2 wt % (pH4) ++ ¹⁾significant discoloration, ²⁾discoloration, ³⁾slightdiscoloration, ⁴⁾no change

The secondary quantitative effect to promote karatolysis was performedby use of at least two different extracts obtained from Cavaliagladiata, Biota orientalis and Coptis chinensis dissolved in 70% ethanolsolution that have been revealed to have efficacy in the firstscreening. Dihydroxy acetone, conventionally used as skin discoloringagent in self-tanning products, was used as a coloring agent. Theexperimental procedure is as follows:

20 persons aged at early twenties-mid thirties were selected and theinner parts of their forearms were selected as a test part. The innerpart was measured in terms of skin brightness before coloring and thencolored with 10% dihydroxy acetone in a hill top chamber. Following 24hr, the colored part was observed with calorimeter and 0.4 ml of each ofsamples comprising at least two extracts dissolved in 70% ethanol with aconcentration of 0.05%, 0.1% and 0.2%, respectively, was applied for 30min twice a day, after which the part applied was observed daily withcolorimeter. The value measured with colorimeter after 24 hr ofdihydroxy acetone application was set 100 and the daily discoloringlevel was converted to %. The evaluation was executed with the periodfor the discolored part's returning to normal color.Inhibitory rate on Hypercornification=[(control group−testedgroup)/control group]×100  Formula 2

TABLE 7 Period for returning according to various concentrations (day)Inhibitory Sample 0.2 wt % 0.1 wt % 0.05 wt % rate (%) Control 19 —Cavalia gladiata:Biota 10 13 16 15.8-47.4 orientalis(50:50) Cavaliagladiata:Coptis 10 12 14 26.3-47.4 chinensis(50:50) Biotaorientalis:Coptis 11 13 15 21.0-42.1 chinensis(50:50) Cavaliagladiata:Biota 9 11 14 26.3-52.6 orientalis:Coptis chinensis(34:33:33)Cavalia gladiata:Biota 9 11 13 31.6-52.6 orientalis:Coptischinensis(70:20:10)

As shown in Table 7, the mixed composition containing at least twoextracts exhibited the inhibitory rate of 47.4-52.6 at a concentrationof 0.2%, demonstrating that the extract of this invention is capable ofturning the skin discoloration by dihydroxy acetone back to normal skincolor in short period of time. The mixed composition comprising at leasttwo extracts showed higher keratolysis effect than that containingsingle extract and in particular, the mixed composition containing threeextracts, Cavalia gladiata, Biota orientalis and Coptis chinensis showedthe highest inhibitory activity against hypercornification.

EXAMPLE 5 Anti-Inflammatory Effect

1) Carrageenan Foot Edema Method Using SD Rat

The extracts from Cavalia gladiata, Biota orientalis, Coptis chinensisand their mixture prepared in Example 1 were intraperitoneallyadministered, respectively, in an amount of 30 mg/kg. After 1 hr, 0.5 mlof 0.1% carrageenan solution was injected into the sole of a back footto induce inflammation. The volume change of rat foot at the time ofinjection of carrageenan and 4-hr after administration was measured andthe Inhibitory rate was calculated according to the Formula 3. Theresults are shown in Table 8.Inhibitory rate (%)=[(1−ΔV _(treated group))/ΔV _(control group))]×100(ΔV: volume change of foot)  Formula 3

TABLE 8 Sample Inhibitory rate (%) Cavalia gladiata 68.8 ± 12 Biotaorientalis 46.0 ± 10 Coptis chinensis 62.3 ± 13 Cavalia gladiata:Biotaorientalis(50:50) 63.1 ± 8  Cavalia gladiata:Coptis chinensis(50:50)70.4 ± 15 Cavalia gladiata:Biota orientalis:Coptis 75.8 ± 9 chinensis(34:33:33) Cavalia gladiata:Biota orientalis:coptis 78.4 ± 10chinensis(70:20:10) Aspirin 35.2 ± 6 2) Balb.c Ear Edema Test

To examine anti-inflammatory effect of the extracts, irritation-inducingsubstances, 5.0% benzalkonium chloride, 2.5% coroton oil and 3000 iu/gretinoic acid (negative control) were dissolved in 70% ethanol solutionand its 0.3 mg/ear was topically applied to mouse ear (6 mice per group)in order to induce edema. At 15 min and 6 hr after induction of edema,respectively, each of the extract and controls (0.3 mg/ear) wastopically applied to mouse ear. After 24 hr of application ofirritation-inducing substances, the applied portion of the mouse was cutoff with a punch and its weight was weighted. The edema extent wasevaluated based on the mean value from the measurements with micrometer.The results are shown in Table 9.

TABLE 9 Inhibitory rate on ear Inhibitory rate on ear Samples weight (%)thickness (%) Cavalia gladiata 49.2 53.8 Biota orientalis 28.8 30.6Coptis chinensis 36.0 38.9 Cavalia gladiata:Biota 34.1 35.3orientalis(50:50) Cavalia gladiata:coptis 44.7 48.0 chinensis(50:50)Biota orientalis:Coptis 32.3 34.6 chinensis(50:50) Cavaliagladiata:Biota 46.0 54.2 orientalis:Coptis chinensis(34:33:33) Cavaliagiadiata:Biota 45.3 58.6 orientalis:Coptis chinensis(70:20:10)Glycyrrhizinic acid 38.4 42.7 Indomethacin 45.6 52.0Inhibitory rate (%)=[(A−B)/A]×100  Formula 4

A: mean value of ear thickness of control group (ear thickness aftertreatment of negative control−ear thickness with no treatment)

B: ear thickness of sample group (ear thickness after treatment ofsample−ear thickness with no treatment)

As shown in Tables 8 and 9, the groups treated with extracts showed theinhibitory activity against edema similar to glycyrrhizinic acid andindomethacin as a positive control. In particular, extract from Cavaliagladiata, and mixed extract from Cavalia gladiata, Biota orientalis andCoptis chinensis exhibited excellent inhibitory effect against edemathat is higher 110-200% than those of negative control solutions.

EXAMPLE 6 Evaluation of Alleviatory Effect on Comedo Using Ear of White

Rabbit

Test system—This test has been generally adopted for evaluation oftreatment. New Zealand white male rabbits aged at 4-month and weighed2-3 kg were purchased and mitigated for 1 week, of which only thehealthy ones determined with naked eye were selected.

Test materials—For test groups, the powderized extracts (0.1% (w/v))from Cavalia gladiata, Biota orientalis, Coptis chinensis and theirmixture dissolved in 70% ethanol solution were used, respectively, 1.0%azelaic acid dissolved in 70% ethanol solution was used as a positivecontrol and 70% ethanol solution per se was used as negative control.

Classification of test systems—Isopropyl myriatate (Sigma) was dailyapplied to both ears of rabbit for 2 weeks for inducing comedo. Rabbitsobserved with naked eye to show typical comedo were selected andclassified to groups each of which was comprised of 6 rabbits inconsideration of comedo severity. After 2 weeks of IPM administration,the appearance of rabbit ear was observed to show severe enlargement ofhair follicle and comedo formation, severe hypercornification ofepidermis and hair follicle and eschar formation and severeinflammation.

Administration method of test materials—Test groups and a positivecontrol to the right ear and a negative control to the left ear ofrabbit were administered once daily for 2 weeks in the amount of 0.5 mland spread with a swab. After 2 weeks, both ears were evaluated.

<Observation with Naked Eye>

The standards for determination with naked eye were classified to 7grades, 0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0. The area of comedo wasevaluated. The period for treatment of comedo was 2-week and theaggravation and alleviation of comedo were determined. The results areshown in Table 10.

<Standards for Determination with Naked Eye>

-   0: no difference compared to control-   0.1: symptoms between 0 and 1-   1: showing congestion, bleeding of capillary tube, slight    cornification and enlargement of hair follicle-   1.5: symptoms between 1 and 2-   2: medium severity of cornification and enlargement of hair follicle-   2.5: symptoms between 2 and 3-   3: excessive severity of cornification and enlargement of hair    follicle, showing typical comedo

TABLE 10 Before After administration administration Inhibitory rateSample (left ear) (right ear) (%) Cavalia gladiata 1.76 ± 0.12 1.21 ±0.08 31.3 Biota orientalis 1.74 ± 0.10 1.44 ± 0.12 17.2 Coptis chinensis1.78 ± 0.09 1.18 ± 0.13 33.7 Cavalia 1.73 ± 0.12 1.12 ± 0.09 35.3gladiata:Biota orientalis(50:50) Cavalia 1.69 ± 0.14 1.14 ± 0.09 32.5gladiata:Coptis chinensis(50:50) Biota orientalis:Coptis 1.62 ± 0.071.09 ± 0.14 32.7 chinensis (50:50) Cavalia 1.76 ± 0.10 1.04 ± 0.08 40.9gladiata:Biota orientalis:Coptis chinensis(34:33:33) Cavalia 1.79 ± 0.091.05 ± 0.11 41.3 gladiata:Biota orientalis:Coptis chinensis(70:20:10)Azelaic acid 1.58 ± 0.12 1.13 ± 0.14 28.5 70% ethanol 1.76 ± 0.10 1.73 ±0.09 1.7<Observation with Image Analysis>

After the observation with naked eye, each test group was euthanizedwith sodium pentobarbital and its both ears were cut out, after whichtheir tissues at basement were cleaved in a size of about 2.5×1.5 cm andimmersed in warm water (50° C.) for 3 min. The epiderms from the tissueswere detached, placed with inner side upward on slide glass, fixed anddried at room temperature. The image was observed with stereoscopicmicroscope (×20) and the number of comedo on tissue and its area wascalculated with image analyzer. The results are shown in Table 11.

TABLE 11 Before administration After (left ear) administration Area of(right ear) comedo/ Area of number comedo/number Inhibitory Sample (cm²)(cm²) rate (%) Cavalia gladiata 0.30 ± 0.02 0.16 ± 0.02 46.7 Biotaorientalis 0.32 ± 0.01 0.23 ± 0.01 28.1 Coptis chinensis 0.30 ± 0.020.17 ± 0.02 43.3 Cavalia gladiata:Biota 0.31 ± 0.02 0.16 ± 0.01 48.4orientalis(50:50) Cavalia gladiata:Coptis 0.29 ± 0.01 0.19 ± 0.01 34.5chinensis(50:50) Biota orientalis:Coptis 0.31 ± 0.02 0.17 ± 0.02 45.2chinensis(50:50) Cavalia gladiata:Biota 0.32 ± 0.01 0.11 ± 0.02 55.4orientalis:Coptis chinensis(34:33:33) Cavalia gladiata:Biota 0.32 ± 0.010.11 ± 0.02 65.6 orientalis:Coptis chinensis(70:20:10) Azelaic acid 0.30± 0.02 0.18 ± 0.02 40.0 70% ethanol 0.31 ± 0.01 0.30 ± 0.02 3.2

As represented in Table 11, the extracts showing higher comedodecreasing ratio (area/count ratio) than azelaic acid (40.0%) as apositive control were Cavalia gladiata (46.7%), Coptis chinensis (43.3%)and composition comprising at least two extracts. In particular, theextract comprising Cavalia gladiata:Biota orientalis:Coptis chinensis(70:20:10) was revealed to show 1.6-fold comedo decreasing ratio higherthan that of a positive control. This experiment demonstrating comedodecreasing ratio represents that results from observation with naked eyeand image analysis are similar each other.

Summarizing the experiments depending on the mechanisms of acnedevelopment, the present extracts were demonstrated to exhibitantimicrobial activity against acne-causing bacteria, P. acnes,inhibitory effect of excess production of sebum, lysis effect of comedo,inhibitory effect of hypercornification and anti-inflammatory effect aswell as safety to skin.

EXAMPLE 7 Evaluation of Irritation to Human Skin

To test skin safety, the extracts of Cavalia gladiata, Biota orientalis,and Coptis chinensis and the mixed extract comprising at least twodifferent oriental medicines that were elucidated to show the treatmentand prevention efficacies against acne was prepared in a form ofemulsion and the human skin irritation test (human patch test) wascarried out by using them. 20 μl of test materials (10% patch base)prepared were added dropwise to a finn chamber, were washed with 70%ethanol and dried, and placed on the test part, the inner part offorearm of 30 healthy persons aged 20-30, followed by fixing withmicroporous tape. After application for 24 hr with patch, the patch wasdetached and the test part was marked with oil pen. The skin responseafter 30 min- and 24 hr-application was observed and evaluated withreferring to the standard as below:

In Table 12, the mean responsiveness is classified to ± (1 point), + (2points), ++ (3 points) and +++ (4 points). The standards fordetermination are grade 1 (no irritation range) ranking below 1, grade 2(slight irritation range) ranking below 3, grade 3 (intermediateirritation range) ranking below 5 and grade 4 (strong irritation range)ranking above 5.

TABLE 12 Mean respon- 24 hr 48 hr siveness Sample ± + ++ +++ ± + ++ +++(n = 30) Grade Acne 4 − − − − − − − 2.00 2 emulsion base Base + 2 − − −− − − − 1.00 1 Cavalia* 0.3 wt % Base + 6 − − − − − − − 2.75 2 Biota**0.3 wt % Base + 4 − − − − − − − 2.00 2 Coptis*** 0.1 wt % Base + 4 − − −− − − − 2.00 2 Cavalia 0.3 wt % + Biota 0.3 wt % Base + 3 − − − − − − −1.50 2 Cavalia 0.3 wt % + Coptis 0.1 wt % Base + 4 − − − − − − − 2.00 2Biota 0.3 wt % + Coptis 0.1 wt % *Cavalia gladiata **Biota orientalis***Coptis chinensis

As shown in Table 12, the acne emulsion base showed irritation level ofsafety range, all oriental medicines except for Cavalia gladiata(grade 1) exhibited irritation level of above grade 2. Coptis chinensiswas tested at a lower concentration due to pigmentation.

EXAMPLE 8 AND COMPARATIVE EXAMPLE 1 Preparation of Emulsion

The mixed extract of Cavalia gladiata, Biota orientalis and Coptischinensis exhibiting the highest comedo lysis and decreasing effects wasformulated in a form of emulsion with ingredients and their amountsdescribed in Table 13. The preparatory procedure is as follows:

TABLE 13 Ingredients Example 8 (wt %) Com. Ex. (wt %)  1. cetearylalcohol 5.00 5.00  2. polysorbate-60 0.80 0.80  3. dimethicone copolymer0.80 0.80  4. cyclomethicone 4.00 4.00  5. hohoba oil 2.00 2.00  6.distilled water Up to 100 Up to 100  7. Canavalia gladiata:Biota 0.50absent    orientalis:Coptis    chinensis(70:20:10)  8. methyl paraben0.20 0.20  9. 1,3-bulylene glycol 8.00 8.00 10. carbomer 0.15 0.15 11.xanthan gum 0.05 0.05 12. polyacrylamide/C13-14 1.20 1.20   isoparaffin/laures-7 13. imidazolydinylurea 0.20 0.20 14. triethanolamine 0.15 0.15 15. perfume 0.10 0.10

The raw materials (ingredients 1-5) present in oil phase were exactlyweighed, introduced into additional oil phase tank and dissolved byheating at 75° C. The raw materials (ingredients 6-9) present in waterphase were weighed and introduced into an emulsifying tank. The rawmaterials (ingredients 10-11) were weighed, wetted with distilled waterand introduced into the emulsifying tank. The ingredients in theemulsifying tank were dissolved with heating at 75° C. The ingredientsof oil phase were introduced into the emulsifying tank under vacuum andemulsified for 5 min at 75° C. using a homogenizer (3500 rpm) and apedal mixer (25 rpm). To the tank, ingredient 12 was added and dispersedfor 3 min. Ingredients 13 and 14 were dissolved and dispersed in smallamount of distilled water and introduced into the tank forneutralization. After defoamation under vacuum and cooling to 50° C.,perfume was added, dispersed and cooled down to 35° C., and theresultant composition was maturated to prepare emulsion.

EXAMPLE 9 AND COMPARATIVE EXAMPLE 2 Preparation of Patch

TABLE 14 Example 9 Com. Ex. Ingredients (wt %) (wt %)  1. Glycerine15.00  15.00   2. Polyarylic acid 2.00 2.00  3. Acrylate copolymer 2.002.00  4. Ammonium hydroxide 0.40 0.10  5. Disodium-EDTA 0.05 0.05  6.Tartaric acid 0.20 0.20  7. Methyl paraben 0.20 0.20  8. Ethanol 1.001.00  9. Glycyrrhizinic acid dipotassium 0.10 0.10 10. Perfume 0.05 0.0511. Cavalia gladiata:Biota 0.50 —    orientalis:Coptis   chinensis(70:20:10) 12. Propylene glycol 5.00 5.00 13. Distilled waterUp to 100 Up to 100

Ingredients 1-6 were weighed, introduced into a dissolving tank andhomogeneously agitated at room temperature to prepare a viscoussolution. Ingredients 7-10 were dissolved at room temperature,introduced into the dissolving tank and homogeneously agitated.Ingredients 11-13 were heated to 60° C., dissolved and introduced inaliquots with small volume, thus giving gel for patch. The gel for patchcomprising the ingredients was homogeneously coated at the amount of 700g/m² on polyester film with siliconized adhesive layer in a size of 1m×1 m by use of coating apparatus with applicator cap, to preparenonporous polyethylene film. The multilayered laminate was cut in a formof circular patch with a diameter of 1.5 cm and introduced into pouchlaminated film paper, low density polyethylene and aluminum, therebyfinally preparing clinical patch.

EXAMPLE 10 Clinical Test with Emulsion and Patch

Example 8 and Comparative Example 1 (test group of 15 persons andcontrol group of 15 persons), and Example 9 and Comparative Example 2(test group of 15 persons and control group of 15 persons) wereevaluated in terms of alleviation effect to acnes. To 60 healthy personsaged at tens-twenties with acne symptoms, the emulsion was topicallyapplied twice daily (in the mornings and evenings) and the patch wasapplied once daily (applied in the evenings and removed in the nextmornings) for 1 month, and the treatment and prevention effects of acnewere evaluated. Prior to test, skin and acne condition of testes werescored and photographed. After 1-month of the application of emulsionand patch, the extent of prevention and treatment of acne was determinedbased on photos and observation with naked eye. The standards fordetermination are found in Table 15.

TABLE 15 Grade Standards for determination of skin conditions 0 Normalcondition 1 Negligible comedo counts or no development of inflammation 2Noticeable comedo counts and size but no development of inflammation 3 Asmall counts of inflammatory papule 4 Crystalline and inflammatorycomedo with small counts and broad distribution 5 Papulosa nodus withsmall counts and size 6 Noticeable counts of inflammatory nodus 7 Highlynoticeable counts of inflammatory nodus 8 Severe crystalline comedo 9Severe vesicula and inflammatory nodus

Grades were determined based on the standards for determination in Table15, where the grade on acne condition of testee was decreased by above 3grades, it was evaluated as excellent efficacy; the grade was decreasedby 1-2 grades, it was evaluated as medium efficacy; the grade was notdecreased, it was evaluated as no efficacy; and the grade was increased,it was evaluated as aggravation. Table 16 represents the clinicalresults of acne prevention and treatment using emulsion and patch ofthis invention with referring to the standards for determination.

TABLE 16 Excellent Medium No Aggra- Adverse Test group efficacy efficacyefficacy vation effect Emulsion Test 12(80%) 2(13.3%) 0 0 1(6.7%) groupControl  0 5(33.3%) 9(60%) 0 1(6.7%) Patch Test 11(73.3%) 4(26.7) 0 0 0group Control  0 6(40%) 9(60%) 0 0

Summarizing the results in Table 16, the emulsion and patch comprisingCavalia gladiata, Biota orientalis and Coptis chinensis exhibit improvedprevention and treatment effects against acne as compared to thosewithout them.

EXAMPLE 11 Toxicity Test

The extracts obtained from Cavalia gladiata, Biota orientalis and Coptischinensis were tested in terms of toxicity as follows: the extractdissolved in dimethylsulfoxide (DMSO) was diluted with water andadministered in the amount of 100 mg/kg to each mouse (10 mice pergroup), followed by observation for 7 days. No mouse was dead.

As described previously, the present topical formulation comprisingextract obtained from at least one oriental medicine selected from thegroup consisting of Cavalia gladiata, Biota orientalis and Coptischinensis, are extremely effective in prevention and treatment of acnesthrough inhibition of the activity of 5α-reductase, antimicrobialactivity against P. acnes, anti-inflammatory action, inhibition ofcomedo formation and keratolysis.

1. A method for treating acne, comprising administering to a subject inneed of such treatment a topical formulation comprising extract obtainedfrom two different kinds of oriental medicine, wherein said extractcomprises 0.01-10 wt % of Cavalia gladiata and 0.01-10 wt % of Biotaorientalis; 0.01-10 wt % of Cavalia gladiata and 0.001-5 wt % of Coptischinensis; or 0.01-10 wt % of Biota orientalis and 0.001-5 wt % ofCoptis chinensis.
 2. A method for treating acne, comprisingadministering to a subject in need of such treatment a topicalformulation comprising extract obtained from three different kinds oforiental medicine, wherein said extract comprises 0.01-10 wt % ofCavalia gladiata and 0.01-10 wt % of Biota orientalis and 0.001-5 wt %of Coptis chinensis.
 3. The method according to claim 1, wherein saidformulation is in the form of emulsion, gel, pack, cosmetic liquid orsoap for cosmetics, ointments or patches for pharmaceuticals.
 4. Themethod according to claim 2, wherein said formulation is in the form ofemulsion, gel, pack, cosmetic liquid or soap for cosmetics, ointments orpatches for pharmaceuticals.
 5. The method according to claim 1 or 2,wherein administration of said topical formulation induces an inhibitoryeffect on 5α-reductase, an antimicrobial activity against acne pathogen,an inhibitory activity to hypercornification, an alleviatory effect oncomedo, or a combination thereof.